The present invention relates to an apparatus and method for high resolution continuous flow electrophoresis, and makes use of a shifted recycle of the solvent/solute flow to achieve high resolution without solute dilution.
Electrophoretic separations take advantage of the differential mobility of solute materials in an external electric field. Solutes will migrate in the field at differing rates which are dependent on parameters such as charge, size, and diffusion coefficient of the solute, and ionic strength of the buffer. If the mobilities of the solutes to be separated are only slightly different, a longer duration of exposure to the electric field will be required to achieve separation. This longer time, however, increases the importance of dispersive effects, and leads to a broadening of the solute containing region leading to solute dilution and a loss of resolution.
Gel electrophoresis is one technique which has been employed for separations. In this technique, dispersive effects are minimized by performing the separation in a gel matrix. Regions of the gel containing the separated solute bands can then be excised, and the solutes recovered from the gel. This technique works well on an analytical scale, but it has obvious limitations which detract from its usefulness on a larger preparative scale.
In response to the need for preparative electrophoretic separations, apparatus have been developed which separate solutes contained within a continuous liquid flow. For example, British Patents Nos. 1,150,722 and 1,186,184 to Philpot describe an annular elecrophoresis chamber through which solutes and solvent flow. An electric field is applied radially, i.e., perpendicular to the solvent flow, causing solute molecules to migrate as concentric rings within the annular chamber. Multiple outlet or collection ports are used to collect the materials flowing out of the chamber in different fractions taken at varying radial distances from the center of the annulus.
Another apparatus for continuous flow electrophoresis utilizes laminar flow of solvent and solutes through a very thin rectangular chamber. Hannig, K., Electrophoresis 3, 235-243 (1982). The electric field causes lateral migration of solutes, and the separated materials are collected through multiple outlet ports at the bottom of the apparatus.
Continuous flow electrophoresis devices of these types have not, however, proven to be suitable candidates for scale-up from a bench-top level. This unsuitability is largely the result of two effects: buoyancy instability as an indirect result of Joule heating creating density inversions within the solvent, and decreased resolution due to convective and electroosmotic dispersion of solute molecules. These difficulties become more pronounced as the electrophoretic apparatus becomes larger and the residence time of ions in the field becomes longer--changes which are associated with preparative separations and those involving difficult-to-separate solutes.
A related technique, isoelectric focusing, can also be used to achieve separation of solutes, particularly of biological molecules. In isoelectric focusing, a pH gradient is imposed upon the solute between the electrodes. A solute molecule migrates in the field until it reaches the point in the pH gradient where it is electrically neutral. This type of separation can also be carried out in a continuous flow apparatus, and may be improved by recycling the flow through the chamber. U.S. Pat. Nos. 4,204,929 and 4,362,612 to Bier describe apparatus for recycle isoelectric focusing (RIEF).
It should be understood, however, that electrophoresis and isoelectric focusing are fundamentally distinct techniques. Electrophoretic separations are rate-driven separation processes in which solute migration continues as long as the electric field is applied. In contrast, isoelectric focusing is an equilibration-process in which solutes only migrate until they reach their characteristic equilibrium point within the pH gradient. For this reason, improvements which are effective for one type of separation technique would not be theoretically expected to improve the other type of technique.